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Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan
ABSTRACT
Unmethylated CpG motifs in bacterial DNA or synthetic oligodeoxynucleotides (ODN) potently stimulate the innate immune system, and they are recognized by Toll-like receptor 9 (TLR9), which is expressed by monocytes/macrophages, dendritic cells, and B cells. However, it is unknown whether alveolar macrophages (AMs) express functional TLR9. To clarify this, we analyzed mRNA expressions of TLRs in murine AMs by real-time polymerase chain reaction, and compared with those in other tissue macrophages and lung antigen-presenting cells. In addition, we determined the sensitivity of these cell populations to CpG-ODN. Interestingly, TLR9 mRNA was almost absent in AMs, but highly expressed in bone marroweCderived macrophages and peritoneal macrophages, whereas TLR2 and TLR4 were present in all macrophage populations. Consistent with the receptor expression, AMs showed no sensitivity to CpG-ODN, whereas other macrophage populations secreted tumor necrosis factor , interleukin 12 p40, and interleukin 6, and enhanced expression of CD40, CD80, and CD86, in response to CpG-ODN. Lung dendritic cells and B cells highly expressed TLR9 mRNA and responded to CpG-ODN. These results indicate selective loss of TLR9 expression in AMs with no sensitivity to CpG-ODN, suggesting that dendritic cells and B cells play a role in the immune response against bacterial DNA in the lung.
Key Words: alveolar macrophages dendritic cells Toll-like receptor 9
Alveolar macrophages (AMs) are highly specialized mononuclear phagocytic cells located in the alveolar space. They are the first immunocompetent cells to encounter inhaled antigens and they therefore play an important role in regulating local immune responses of the lung (1). Furthermore, AMs have been shown to augment local inflammation by producing a variety of inflammatory mediators, such as cytokines, chemokines, and reactive oxygen intermediates, in response to inhaled particles or microbes (1eC3). Under such conditions, AMs enhance their bactericidal/cytotoxic capacity, which leads to the protection against pathogens. Because AMs constitute the first-line defense against inhaled pathogens, they are likely to highly express the receptors of conserved pathogen-associated molecular patterns (PAMPs), such as bacterial DNA, LPS, and lipoteichoic acid (LTA) (4eC6). In vertebrates, a set of distinct receptors has been developed to specifically recognize PAMPs, and signals through PAMP receptors can stimulate the host immunocompetent cells to activate the innate immune system (7, 8). Recent advances in this area have highlighted a crucial role of Toll-like receptors (TLRs), type I transmembrane proteins, which represent a newly recognized family of PAMP receptors (9eC11). However, little is known about TLR expression and its function in AMs.
Bacterial DNA can potently stimulate the innate immune system in vertebrates (12, 13). This immunostimulatory effect depends on unmethylated CpG dinucleotides within specific flanking bases (CpG motif) present at high frequency in bacterial but not vertebrate DNA (14, 15). CpG motifs of bacterial DNA or synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODN) have been shown to stimulate immunocompetent cells, including macrophages/monocytes, dendritic cells (DCs), and B cells, resulting in cellular activation and production of Ig and cytokines/chemokines (16eC23). This response confers protection against a variety of pathogens containing CpG motifs, and plays a crucial role in linking innate and adaptive immunities. These CpG motifs are recognized by TLR9, which is expressed primarily by monocytes/macrophages, DCs, and B cells (17, 24). Because a variety of microbes invade the lung, this CpG recognition system must have a pivotal role in the innate immunity of this organ. Furthermore, several studies have demonstrated that intrapulmonary CpG-ODN administration elicits local interleukin-12 (IL-12) production, which drives a helper T Type 1 (Th1) immune response in the lung (25, 26). However, it is not fully determined what types of cells in the lung express TLR9 and/or respond to bacterial DNA, although the most likely candidates are AMs because of their location and highly phagocytic function.
The present study was conducted to clarify TLR expression in AMs and its sensitivity to CpG-ODN. We analyzed mRNA expression of TLRs, including TLR2, TLR4, and TLR9, using quantitative real-time polymerase chain reaction (RT-PCR) in murine AMs, and compared these TLR expressions with those of other origin macrophages: bone marroweCderived macrophages (BMs); peritoneal macrophages (PMs); and other lung antigen-presenting cells (APCs), such as lung DCs and B cells. In addition, we evaluated the sensitivity of these cell populations to CpG-ODN. Unexpectedly, we found that TLR9 mRNA was almost absent in AMs, but highly expressed in BMs, PMs, lung DCs, and B cells. On the other hand, TLR2 and TLR4 were present in all of the cell populations. Consistent with the receptor expression, AMs showed no sensitivity to CpG-ODN, whereas other cell populations were activated by CpG-ODN in terms of cytokine production and/or expression of immunostimulatory molecules. The present data indicate that AMs are distinct from other macrophages regarding TLR9 expression and sensitivity to CpG-ODN, suggesting that lung DCs and B cells play a role in response to bacterial DNA in the lung.
METHODS
Animals
Experiments were performed on 10-week-old male BALB/c mice (Nippon SLC, Shizuoka, Japan) housed in a pathogen-free environment throughout the experiment.
Reagents
The CpG-ODN (ODN 1668; TCC ATG ACG TTC CTG ATG CT) and the noneCCpG-ODN (ODN 1720; TCC ATG AGC TTC CTG ATG CT) (19) were synthesized by Bex Co. Ltd. (Tokyo, Japan) and were fully phosphorothioated (see online supplement for details). LPS and LTA were purchased from Sigma (St. Louis, MO).
Preparation of Macrophages
AMs were obtained by whole lung lavage. BMs were grown according to the standard protocols (27, 28). PMs were elicited by peritoneal injection of 3% Brewer's thiogycollate medium (Sigma). Purities of AMs, BMs, and PMs were routinely greater than 98% (see online supplement for details). CD69 antigen, which is one of the activation markers, was not detected in these three macrophage populations (data not shown).
Isolation of Lung DCs and B Cells
To prepare lung DCs and B cells, we used enzymatic digestion and immunomagnetic cell sorting. The digested cells were incubated for 90 minutes at 37°C, and nonadherent cells were then harvested. Of these cells, B cells were sorted using a magnetic cell-sorting system (Miltenyi Biotech, Gladbach, Germany) with anti-CD45R mAb-conjugated magnetic beads. The resulting adherent cells were then also incubated overnight. After overnight incubation, loosely adherent and floating cells were harvested. To purify DCs, CD45R-negative and MHC Class 2eCpositive cells were sorted by the magnetic cell-sorting system. The sorted B-cell population was more than 97% pure, and DC preparation was more than 95% as determined by their morphologic and phenotypic characteristics (see online supplement for details).
RT-PCR
The expression of TLR mRNA was analyzed by quantitative RT-PCR amplification. Total RNA was isolated from AMs, BMs, PMs, lung DCs, and B cells by the acid guanidinium thiocyanate-phenol-chloroform extraction method. Parameter-specific primer sets optimized for the LightCycler (Roche Applied Science, Indianapolis, IN) were purchased from Nippon Gene Research Center (Sendai, Japan). Gene-specific OGN primers were designed using the published sequence of mouse cDNAs for TLR2, TLR4, TLR9, and -actin obtained from GenBank. The PCR was performed with the LightCycler FastStart DNA SYBR Green I kit (Roche Applied Science) according to the manufacturer's protocol. The copy number of the different TLRs was normalized by the housekeeping gene -actin and is presented as the number of transcripts per 104 copies of -actin (see online supplement for details).
Cytokine Assays
The concentrations of IL-6, tumor necrosis factor (TNF-), and IL-12 p40 were measured using ELISA kits from R&D Systems (Minneapolis, MN; see online supplement for details).
Flow Cytometry
For measuring surface expression of immunostimulatory molecules, cells were analyzed with an EPICS XL flow cytometer (Beckman Coulter, Fullerton, CA) after 2-day stimulation of CpG-ODN, LPS, or LTA. The cells were incubated with the appropriately diluted fluorescein isothiocyanateeCconjugated antieCI-Ad mAb (Miltenyi Biotech), phycoerythrin-conjugated anti-CD11c mAb, and biotin-conjugated anti-CD45R, anti-CD8, anti-CD11b, anti-CD40 (BD PharMingen, San Diego, CA), anti-CD80, anti-CD86 (Beckman Coulter), or anti-DEC205 mAb (Diagnostic Products Corp., Nauheim, Germany), followed by incubation with streptavidin-conjugated Quantum Red (Sigma).
Effect of LPS on TLR9 Expression in AMs
For in vitro stimulation, AMs were treated with 1 mg/ml of LPS for 6 to 24 hours. For in vivo stimulation, mice were injected intratracheally with either 1 mg/kg of LPS in phosphate-buffered saline (PBS) or PBS alone. At 24 hours after injection, AMs were recovered. TLR9 mRNA expression was measured in those AMs by RT-PCR (see online supplement for details).
Statistical Analysis
One-way analysis of variance was used for comparisons of groups. The Dunnett post hoc test was used for comparisons of each group. p Values less than 0.05 were considered significant. All data are expressed as mean ± SEM.
RESULTS
Expression of TLR mRNA in AMs, PMs, and BMs
We used quantitative RT-PCR to measure the expression of TLR transcripts, including TLR2, TLR4, and TLR9 in AMs and compared these mRNA expressions with those of other macrophages, BMs and PMs. As shown in Figure 1A, the transcripts of TLR2 and TLR4 were readily detected in all three macrophage populations. The levels of TLR2 mRNA were highest in BMs (Figure 1B). The expression of TLR4 mRNA was significantly higher in PMs and BMs than in AMs (Figure 1C). Regarding TLR9, strikingly, AMs expressed only negligible levels of TLR9 mRNA, whereas BMs and PMs showed strong expression of its transcript (p < 0.001 and p < 0.005, respectively, compared with AMs; Figure 1D). The levels of TLR9 mRNA were threefold higher in BMs than in PMs (p < 0.005; Figure 1D). Collectively, the RT-PCR analysis proved selective loss of TLR9 mRNA expression in AMs, suggesting the distinctive TLR expression patterns of AMs from those of other macrophages.
Response of AMs, PMs, and BMs to CpG-ODN
To test the sensitivity of macrophages to CpG, we assessed cytokine production and expression of immunostimulatory molecules by three macrophage populations in response to CpG-ODN. The immunostimulatory ODN stimulated PMs and BMs to secrete a large amount of these cytokines (Figure 2). IL-6 was secreted from PMs and BMs in a dose-dependent manner, and TNF- production from PMs was also dose-dependent. The concentration of IL-12 p40 in PMs and BMs, and that of TNF- in BMs, reached a peak when treated with 0.3 e CpG-ODN. The amounts of cytokine production were much larger from BMs than from PMs. In contrast, AMs failed to produce IL-6, TNF-, or IL-12 p40 in response to CpG-ODN, even at the highest dose (1.0 e). NoneCCpG-ODN did not stimulate any macrophage populations to secrete the cytokines tested. As to the sensitivity of AMs to other TLR ligands, LPS induced AMs to secrete a large amount of IL-6 and TNF-, whereas LTA stimulated them to produce TNF-, but not IL-6. CpG-ODN also enhanced the expressions of CD40, CD80, and CD86 in BMs and PMs, but not in AMs (Figure 3). NoneCCpG-ODN did not stimulate any macrophage populations to increase the surface expression of those immunostimulatory molecules. In terms of the upregulation of the immunostimulatory molecules, BMs had sensitivity to LPS and LTA, whereas PMs had sensitivity only to LTA. LTA or LPS did not enhance these molecule expressions on AMs. Taken together, these data indicated that CpG responsiveness correlated with TLR9 expression in macrophages, and that AMs impaired the sensitivity to CpG.
Expression of TLR mRNA in Lung APCs
Because AMs were shown to fail to respond to CpG-ODN in accord with their negligible expression of TLR9, the question arose what types of cells express TLR9 and respond to bacterial DNA in the lung. To clarify this, we examined TLR expression in other lung APCs, DCs, and B cells. Purified lung DCs were negative for CD8 and B220 (data not shown), indicating that these were myeloid lineages. RT-PCR analysis revealed that lung DCs and B cells expressed considerable amounts of TLR9 transcripts (Figures 4A and 4D). The levels of TLR9 mRNA expression were much higher in B cells than in DCs. In contrast, TLR2 and TLR4 mRNA were significantly higher in AMs than in lung DCs and B cells (Figures 4B and 4C).
Response of Lung APCs to CpG-ODN
We further examined the CpG responsiveness of lung DCs and B cells. On stimulation with CpG-ODN, lung DCs secreted large amounts of IL-6, TNF-, and IL-12 p40 in a dose-dependent manner, whereas B cells secreted only IL-6, but not TNF- or IL-12 p40 (Figure 5). CpG-ODN also markedly upregulated expressions of CD40, CD80, and CD86 in lung DCs (Figure 6). Control ODN failed to stimulate DCs and B cells to produce cytokines and enhance the expression of the immunostimulatory molecules.
Effect of LPS on TLR9 Expression of AMs
In vitro, LPS treatment did not significantly increase TLR9 mRNA expression in AMs (Figure 7). Intrapulmonary administration of LPS induced a marked increase in the numbers of the total cells and neutrophils in the bronchoalveolar lavage fluid (data not shown), but failed to induce TLR9 mRNA expression in AMs. Taken together, these data suggest that LPS does not affect TLR9 expression in AMs.
DISCUSSION
The present study examined TLR expression in AMs and its sensitivity to CpG-ODN, and compared it with those of macrophages obtained from different tissues and other lung APCs. Strikingly, AMs showed negligible expression of TLR9 and no sensitivity to CpG-ODN, whereas other macrophages, PMs and BMs, strongly expressed TLR9 and responded well to CpG-ODN. Among the lung APCs, TLR9 was highly expressed on DCs and B cells, both of which could be stimulated by CpG-ODN. Collectively, these results highlight impaired TLR9 expression in AMs compared with other macrophages, suggesting that lung DCs and B cells play a role in the immune response against bacterial DNA.
Because the lung is continually exposed to foreign antigens, such as pathogens, PAMP receptors that recognize conserved microbial structure play an important role in activating the innate immunity (8, 29). Among these PAMP receptors, TLR9 specifically recognizes CpG motifs derived from bacterial DNA as danger signals, and ligand-activated TLR9 potently stimulates a wide variety of immune responses, including innate, humoral, and cell-mediated immunity (24, 30). Thus, this CpG recognition system by TLR9 is involved in the linkage between innate and acquired immunity in the lung (10, 11). To date, however, it remains to be determined what types of lung cells respond to bacterial DNA in conjunction with TLR9 expression. We hypothesized that AMs may be the primary cells responding to CpG motifs, because they are situated at the first-line defense in the lower respiratory tract with potent phagocytic capacity (1). However, the present study clearly demonstrated that AMs expressed little TLR9 mRNA, resulting in failure to respond to CpG-ODN. This impairment of TLR9 expression as well as no sensitivity to CpG motifs are a distinctive feature of AMs compared with other origin macrophages, because TLR9 was highly expressed in PMs and BMs, both of which were activated by CpG-ODN. Regarding the other TLRs tested, TLR2 and TLR4 were present in AMs, and the levels of their expression in AMs were comparable to those of PMs. Taken together, these results suggest that selective loss of TLR9, but not TLR2 or TLR4, is a characteristic TLR expression pattern of AMs. Even when stimulated in vitro and in vivo by LPS, TLR9 expression was not significantly upregulated in AMs, suggesting that AMs do not express TLR9 even under inflammatory conditions in the lung, although there is still the possibility that another specific stimulus can induce its expression in AMs.
The reason for the loss of TLR9 expression in AMs is not clear. AMs are terminally differentiated mononuclear phagocytes that have adapted themselves to particulate environments (i.e., alveolar spaces) (31, 32). This differentiation process depends greatly on the local environment, such as the cytokine milieu in the lung (33eC35). To date, it has been reported that AMs have distinctive characteristics compared with other tissue macrophages (36). For example, recent studies have emphasized the suppressive regulatory role of AMs in the local immunity of the lung (2, 3). Unlike macrophages from other tissues, AMs obtained from rodent lungs were shown to suppress the proliferation of antigen- or mitogen-stimulated T cells. More recently, we showed that human AMs highly expressed peroxisome proliferator-activated receptor , or PPAR, which plays an antiinflammatory role through inhibiting cytokine production and increasing CD36 expression together with the enhanced phagocytosis of infiltrating neutrophils (37). Such accumulating evidence suggests that AMs have an important role in maintenance of homeostasis in the lung by inhibiting and/or resolving elicited inflammation in response to inhaled particles. In this context, the particular environment in the lung may diminish TLR9 expression in AMs, resulting in regulation of excessive reaction against bacterial DNA. Further study will clarify the mechanism and/or implication of the impaired TLR9 expression in AMs in the immunity of the lung.
Because AMs did not express functional TLR9, the new question was raised of which APCs bear this receptor in the lung. To clarify this, we next examined TLR9 expression and its sensitivity to CpG-ODN in purified lung DCs and B cells. Lung DCs obtained by enzymatic digestion and immunomagnetic cell sorting were major histocompatibility class II+/CD11c+/CD8eC/B220eC, indicating that they were of myeloid origin (38). We found that both lung DCs and B cells strongly expressed TLR9, which was much higher in B cells than in DCs. Unlike human TLR9 (39), mouse TLR9 expression has been reported to be not restricted to plasmacytoid DCs, and more broad on other DC subsets (17, 40). Our result that lung DCs, which were phenotypically myeloid DCs, expressed TLR9 was consistent with those of the previous reports. Interestingly, on stimulation with CpG-ODN, lung DCs produced large amounts of cytokines, including IL-12 p40, IL-6, and TNF-, whereas lung B cells secreted only IL-6. Furthermore, CpG-ODN also markedly enhanced expression of CD40, CD80, and CD86 in lung DCs, but the same ODN increased only CD40 expression, to a moderate extent, in B cells. Thus, lung DCs and B cells have functional TLR9 and sensitivity to CpG-ODN, but their profiles of cytokine production and upregulated immunostimulatory molecules differ. Collectively, these results suggest that lung DCs and B cells, but not AMs, can respond to inhaled bacterial DNA in the lung.
Several studies have demonstrated that CpG-ODN exposure could prevent allergic airway inflammation and airway hypersensitivity reaction, suggesting a potential therapeutic application of CpG-ODN in asthma (41). Recently, local production of IL-12 was shown to be critical for this prevention (42). In this context, the present study clearly indicates that lung DCs are the potent source of IL-12 induced by CpG-ODN. Inconsistent with our data, Choudhury and coworkers (25) reported that AMs were the primary IL-12eCproducing cells in response to CpG-ODN exposure. They showed that CpG-ODN was internalized in AMs, thereby stimulating AMs to secrete IL-12 p40. In Choudhury and coworkers' study, however, AMs were shown to secrete very small amounts of IL-12 p40 (30eC40 pg/ml), even when stimulated with a high dose of CpG-ODN (2 e) in vitro. In contrast, the present study demonstrated that lung DCs produced much higher amounts of IL-12 p40 (> 6,000 pg/ml) in response to a lower concentration of CpG-ODN (0.3 e), whereas AMs secreted negligible amounts. Taken together, these data suggest that lung DCs, but not AMs, are primarily responsible for local IL-12 production elicited by inhaled bacterial DNA. In addition, Choudhury and coworkers (25) also demonstrated that CpG-ODN and noneCCpG-ODN were equally internalized and colocalized in AMs, but did not prove TLR9 expression in AMs nor ligation between internalized CpG-ODN and TLR9. Although the possibility cannot completely be ruled out that AMs express very low levels of functional TLR9, our data provide evidence that its role in AMs, if any, is likely to be marginal in terms of cytokine production and surface expression of immunostimulatory molecules.
In conclusion, our results indicate the selective loss of TLR9 expression in AMs with no sensitivity to CpG-ODN, which is a distinctive feature of AMs among macrophage populations. Alternatively, lung DCs and B cells express functional TLR9, suggesting that these APCs are the primary cells recognizing and responding to bacterial DNA in the lung. These data provide important knowledge that contributes to the further understanding of the recognition system of PAMPs in the lung.
Acknowledgments
The authors thank Dr. Uchijima for helpful advice and for providing the L929 medium.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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